P38 Kinase, SGK1 and NF-κB Dependent Up-Regulation of Na+/Ca2+ Exchanger Expression and Activity Following TGFß1 Treatment of Megakaryocytes

Transforming Growth Factor β1 (TGFβ1) plays an important role in the maturation of megakaryocyte and formation of platelets. TGFβ1 can up-regulate Ca2+ entry through store operated Ca2+ entry (SOCE) and on the contrary, it can up-regulate Ca2+ exclusion by upregulating the activity of Na+/Ca2+ exchangers. TGFβ1 first enhances the increase of intracellular Ca2+ triggered by the release of Ca2+ from intracellular stores, then it enhances the subsequent decline of [Ca2+]i. The mechanism of action, by which TGFβ1 up-regulates SOCE, is based on a signalling pathway requires the activation of p38 MAP Kinase, Serum & Glucocorticoid inducible Kinase (SGK1), and Nuclear Factor κB (NFκB). On the other hand, the mechanism of action, by which TGFβ1 upregulates Na+/Ca2+ exchangers remained unidentified, as well as the specific Na+/Ca2+ exchanger isoforms involved in the process of up-regulation. The present study aimed to identify, whether TGFβ1 influences the expression and activity of K+-independent (NCX) and K+-dependent (NCKX) Na+/Ca2+ exchangers, and aimed also to explore the signalling involved. Methods: In human megakaryocytic cells (MEG01), Fura-2 fluorescence was utilized to observe cytosolic Ca2+ activity [Ca2+]i. The activity of Na+/Ca2+ exchanger was studied by observing the rise in [Ca2+]i resulting from changing the extracellular solution from a solution with 0 mM Ca2+ and 130 mM Na+ to a solution with 2 mM Ca2+ and 0 Na+. For analysis of NCX, the concentration of K+ was 0 mM. For analysis of NCKX, the concentration of K+ was 40 mM. In order to quantify transcription levels of NCX/NCKX isoform, RT-PCR was applied. Results: TGFβ1 (60 ng/ml, 24 h) was found to increase significantly the transcription levels of certain isoforms of NCX/NCKX including: NCX1, NCKX1, NCKX2 and NCKX5. Additionally, the activity of NCX and NCKX was shown to be increased significantly in the presence of TGFβ1 (60 ng/ml, 24 h). Skepinone-L (1 μM), a p38 MAP Kinase inhibitor, caused a significant downregulation of the effect of TGFβ1 on both transcription levels and activity of NCX and NCKX. GSK-650394 (10 μM), an inhibitor of SGK1, and Wogonin (100 μM), and inhibitor of NFκB, caused a significant downregulation of the effect of TGFβ1 on the activity of NCX and NCKX. Conclusions: P38 MAP Kinase, SGK1 and NFκB are involved in the signaling pathway by which TGFβ1 increases the activity of Na+/Ca2+ exchanger and the transcription levels of NCX1, NCKX1, NCKX2, and NCKX5